Takehito Igarashi

Overcoming graft rejection in heavily transfused and allo-immunised patients with bone marrow failure syndromes using fludarabine-based haematopoietic cell transplantation

Allogeneic haematopoietic cell transplantation (HCT) can cure a variety of non‐malignant haematological disorders. Although transplant outcomes for selected patients with severe aplastic anaemia (SAA) and paroxysmal nocturnal haemoglobinuria (PNH) have improved, older age, allo‐immunisation from transfusions, prior immunosuppressive therapy and a prolonged time from diagnosis to transplantation are associated with worse outcome. Because of its potent immunosuppressive effects, we investigated a fludarabine‐based non‐myeloablative conditioning regimen in patients with transfusion‐dependent non‐malignant haematological disorders at increased risk for graft rejection with conventional transplant conditioning. Twenty‐six patients with transfusion dependent/anti‐thymocyte globulin (ATG)‐refractory SAA, PNH or pure red cell aplasia underwent HCT from a human leucocyte antigen (HLA)‐compatible relative. Transplant conditioning consisted of cyclophosphamide (120 mg/kg) and fludarabine (125 mg/m2) with or without ATG. Ciclosporine, alone or combined with mycophenolate mofetil or methotrexate, was used as graft‐versus‐host disease (GVHD) prophylaxis. All patients achieved durable engraftment and transfusion‐independence. Twenty‐four of 26 patients are alive at a median of 21 months following transplantation. Although a high cumulative incidence of acute (65% grades II–IV, 54% grades III–IV) and chronic GVHD (56%) was observed, only one patient died from transplant‐related causes (cumulative incidence 7%). These data show that HCT following fludarabine‐based non‐myeloablative conditioning results in durable engraftment and excellent survival in SAA and PNH patients at high risk for graft rejection.

Suicide Gene Therapy for Chemically Induced Rat Bladder Tumor Entailing Instillation of Adenoviral Vectors

The efficacy of an in vivo gene therapy protocol making use of an adenoviral vector in the treatment of bladder cancer was examined. Bladder tumors were induced in rats by oral administration of BBN (N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine). Histologically, such tumors resemble those seen in human bladder cancer, and the cells can be selectively transduced using adenoviral vectors. The therapeutic protocol thus entailed instillation of an adenoviral vector containing the HSV‐tk suicide gene into rat bladder followed by a regimen of intraperitoneal ganciclovir (GCV) injections. Histological examination after a short‐term GCV regimen (3 days) revealed marked vacuolization of the tumor cells. Moreover, TUNEL assays showed that the cytotoxic reaction was mediated by apopto‐sis. Following a long‐term GCV regimen (14 days), tumor growth was significantly inhibited and glandular metaplasia was observed. This is the first report demonstrating the efficacy of in vivo suicide gene therapy in a chemically induced transitional cell carcinoma like that seen in most human bladder cancer. Intravesical instillation is already a well established clinical technique. Our findings indicate that now there is a strong potential for its incorporation into new and useful gene therapies aimed at the treatment of human bladder cancer.

Identical germline mutations in the TMEM127 gene in two unrelated Japanese patients with bilateral pheochromocytoma

Recently, TMEM127 was shown to be a new pheochromocytoma susceptibility gene; this is consistent with its function as a tumour suppressor gene (Journal of Clinical Endocrinology and Metabolism, 2009, 94, 2817). Most pheochromocytomas arise from the adrenal medulla, and in approximately half of the cases, the tumours are bilateral (Journal of Clinical Endocrinology and Metabolism, 2009, 94, 2817; Journal of the American Medical Association, 2004, 292, 943; Human Mutation, 2010, 31, 41; Science, 2009, 325, 1139). The aim of the present study was to determine whether TMEM127 mutations are involved in the pathogenesis of pheochromocytomas/paragangliomas in Japanese subjects.

In situ Gene Transfer and Suicide Gene Therapy of Gastric Cancer Induced by N-Ethyl-N′-nitro-N-nitrosoguanidine in Dogs

Gene therapy could potentially revolutionize the treatment of gastrointestinal (GI) tract cancer. The aim of this study was to establish a practical method of gene transfer which would be applicable to human gastric cancer. Retrovirus or/and adenovirus vectors carrying the lacZ marker gene were transferred in situ by needle through an endoscopic biopsy channel into primary gastric cancer in six male beagle dogs that had been treated with N‐ethyl‐N′‐nitro‐N‐nitrosoguanidine (ENNG). In addition, an adenovirus vector carrying the herpes simplex virus thymidine kinase (Ad.CAGHSV‐TK) gene was introduced in situ into cancer tissues in the stomach of three dog, and the animals were treated with intravenous ganciclovir (GCV). Retrovirus‐producing cells which expressed the lacZ gene were specifically localized to the injection site in the stomach. The lacZ gene was more widely transferred into the tumor by the adenovirus vector than by retrovirus‐producing cells. Improvement of the needle used for gene transfer and the use of multiple injections per tumor led to more diffuse transfer of the vector into the tumor. The Ad.CAG lacZ gene was also transferred into regional lymph nodes of the stomach. Moderate to diffuse degeneration of the primary cancer tissues of the stomach was found after Ad.CAGHSV‐TK/GCV gene therapy. Moreover, almost complete tissue degeneration was observed in the regional lymph nodes of the stomach. An adverse effect of HSV‐TK/GCV gene therapy was acute hepatotoxicity, which was not found after Ad.CAG lacZ gene transfer, but was found after high‐titer Ad.CAGHSV‐TK gene transfer followed by GCV. These findings suggest that in situ gene transfer of a suicide gene followed by prodrug treatment may be applicable not only to primary tumors, but also to lymph node metastases of gastric cancer, though further study of both beneficial and adverse effects is required before clinical usage.

Significance of a solid component in the papillary thyroid carcinoma associated with clinicopathological parameters.

Solid variant of papillary thyroid carcinoma (SVPTC) is characterized by a solid component (SC) involving more than 50% of the tumour with the preservation of the classical cytological features of papillary thyroid carcinoma (PTC). However, the clinical significance of SC in PTC has been rarely examined. Herein, we investigated retrospectively the clinicopathological features of PTC with various degrees (10–85%) of SC (PTCSC).

Expression of MRP1 and ABCG2 is associated with adverse clinical outcomes of papillary thyroid carcinoma with a solid component

Solid variant of papillary thyroid carcinoma (PTC) is characterized by a solid component (SC) retaining classical cytological features of PTC. Despite some controversies, PTC with SC (PTCSC) cases have poor prognosis compared with well-differentiated PTC. We investigated if cancer stem cells (CSCs) may have a role in pathogenesis of PTCSC. PTCSC tumors (n=27) were histologically represented by a mixture of papillary component (PC) and varying degrees of SC involving 10% to 85% of the tumor. Immunohistochemical expression of CSC markers ABCG2 and MRP1, and HBME1 and CK19 was compared between SC and PC within each tumor in association with clinicopathological parameters. ABCG2 and MRP1 were highly expressed in SC, whereas their expression was limited or absent in PC (P=.04 and .002, respectively). In contrast, expression of HBME1 and CK19 appeared higher in PC than in SC (P=.08 and .02, respectively). Higher expression of ABCG2 was associated with higher incidence of large-sized SC (P=.01). Higher expression of MRP1 was associated with higher incidence of lymphovascular invasion (P=.049). Higher expression of ABCG2 and MRP1, and lower expression of CK19 in SC were associated with higher tumor recurrence rate (P=.02, .01, and .02, respectively), and shorter disease-free survival (P<.001 for all the variables). Our findings indicate that the tumor cells harboring CSC-like characteristics in SC could contribute to the pathogenesis of PTCSC and might account for the poor disease prognosis.

Fine needle aspiration cytology of the papillary thyroid carcinoma with a solid component: A cytological and clinical correlation

Solid variant of papillary thyroid carcinoma is a rare subtype of papillary thyroid carcinoma (PTC) containing a solid component (SC), and thus its cytological and clinicopathological features remain elusive. We examined fine needle aspiration (FNA) cytological features of PTC with variable degrees of SC (20‐80% of the tumor)(PTCSC) in comparison to well‐differentiated PTC (WPTC).

CQ24. Does Total Thyroidectomy with Radioactive Iodine Ablation and TSH Suppression Therapy Improve Patient Prognosis with Widely Invasive Follicular Carcinoma Compared to Only Limited Thyroidectomy

The widely invasive type is more likely to show recurrence and distant metastasis than the minimally invasive type, resulting in a poorer prognosis (see CQ22). In this CQ, how to manage the widely invasive type diagnosed on pathological examination is investigated.

CQ22. Does Classification of Follicular Carcinoma According to the Degree of Invasiveness (Widely Invasive and Minimally Invasive Types) Reflect the Prognosis

Preoperative diagnosis of follicular carcinoma is difficult and most diagnoses are established on pathological examination. If it is possible to predict the prognosis by pathological classification, it would be beneficial in determining the treatment strategy after initial surgery. Even when the invasion can only be detected under the microscope, if the invasion covers a grossly large area, the tumor is classified as widely invasive.

CQ21. Is It Possible to Diagnose Follicular Carcinoma on Preoperative FNA or Intraoperative Frozen Section Diagnosis

Follicular carcinoma is defined as “a malignant tumor arising from follicular epithelial cells lacking the typical findings of the nuclei for papillary carcinoma,” but the diagnosis is extremely difficult. The follicular structure of follicular carcinoma is often very well differentiated, to the extent that it is difficult to discriminate from that of the normal thyroid. Generally, structural atypia, cellular atypia and nuclear atypia are important findings to establish malignant tumors. These atypia are clearly observed in FNA specimens of most papillary carcinomas and the treatment strategy for papillary carcinoma can be determined preoperatively. However, follicular carcinoma can be diagnosed only when the presence of capsular and/or vascular invasion is identified on pathological examination, requiring the tissue specimens from a number of lesions, including the whole capsule. A follicular tumor with these pathological findings is diagnosed as follicular carcinoma and in their absence as follicular adenoma. The above-mentioned atypia is not included in the diagnostic criteria of follicular carcinoma.